Vitamin B.sub.12 is an organo-cobalt compound of molecular weight 1355.42. Vitamin B.sub.12 is not manufactured in the human body and must be ingested in order to meet our nutritional needs. There are several endogenous transport proteins in the body which aid in the absorption of B.sub.12 from food. Included among these are haptocorrins in the saliva which bind B.sub.12 as it is released during digestion. Another transport protein is intrinsic factor, found in the intestinal tract, which transport B.sub.12 across the intestinal mucosa into the blood stream. Further transport proteins are the transcobalamins in the blood stream, which bind B.sub.12 and deliver it to the tissues of the body that require it. In the blood stream B.sub.12 is present as hydroxy-, methyl- or adenosylcobalamin. These derivatives are formed by replacing the axial cyano group of B.sub.12 with the respective functional group. The terms "B.sub.12 " and "vitamin B.sub.12 " as used herein are intended to encompass all of these derivatives collectively.
Since part of the B.sub.12 in serum is bound to transcobalamin binding proteins, any assay which measures B.sub.12 must first pretreat the sample to release the B.sub.12 from these proteins. A further interfering protein is anti-intrinsic factor antibody. This antibody when present, as it is in samples of patients suffering from certain states of pernicious anemia, must also be denatured during pretreatment since such antibody will interfere with any competitive assay which utilizes intrinsic factor as the B.sub.12 -binding protein. A detailed discussion of these problems is found in Vitamin B.sub.12, B. Zagalak and W. Friedrich, editors (Walter de Gruyter & Co., Berlin, 1979); B12 Volume 1: Chemistry and B.sub.12 Volume 2: Biochemistry and Medicine, D. Dolphin, editor (John Wiley & Sons, New York, 1982).
To date the most common assays for Vitamin B.sub.12 are radioimmunoassays (RIA). Most of these methods utilize intrinsic factor bound to a solid phase and B.sub.12 radio-labeled with .sup.57 Co competing with B.sub.12 in the sample. The sample is pretreated either by a "boil" method or a "no-boil" method. The boil method entails boiling the sample in the presence of a thiol compound at pH 9.3 to denature the B.sub.12 -binding proteins and anti-intrinsic factor antibodies. Potassium cyanide is also included in the pretreatment solution to convert all forms of B.sub.12 to cyanocobalamin. In the no-boil method, sodium hydroxide and a thiol compound are added to denature the endogenous binding proteins and anti-intrinsic factor antibodies. Potassium cyanide is again added as in the boil method. After pretreatment, a neutralizer is added to bring the pH down to 9.3. This system has been investigated in detail by Allen in U.S. Pat. Nos. 4,188,189, 4,351,822 and U.S. Pat. No. 4,451,571, which note the need to use purified intrinsic factor in a radio dilution assay and cobinamide in addition to alkali and thiol for the no-boil sample pretreatment.
While enzyme immunoassays have been known since the early 1970's--see, for example, Engvall, E., and Perlmann, P., Immunochem. 8:871 (1971)--the sensitivity of the method has only recently been sufficiently improved to permit B.sub.12 to be measured at clinically significant levels (i.e., pg/mL). Such an improvement was first published by Bachas, L. G., Tsalta, C. D., and Meyerhoff, M. E., in Biotechniques 4:42-55 (1986), who describe a B.sub.12 enzyme immunoassay which utilizes intrinsic factor attached to beads, with B.sub.12 -glucose-6-phosphate dehydrogenase as the probe.
Since the Bachas, et. al. publication, several abstracts have appeared relating to B.sub.12 immunoassays. One of these, Wang, C. -C., Charlton, R. R., "An Enzymometric Assay for Vitamin B.sub.12 Using Magnetic Particles as Solid Support," Clin. Chem. 33:963 (1987), described an assay which uses B.sub.12 coupled to chromium dioxide, intrinsic factor-.beta.-galactosidase and .alpha.-nitrophenyl-.beta.-D-galactoside as substrate. In May 1989, a poster was presented by Dworschack, et. al. at a CLAS meeting in Los Angeles, pertaining to an assay which employs the CEDIA technology with intrinsic factor as the B.sub.12 -binding protein and B.sub.12 -enzyme donor (enzyme donor is a genetically engineered fragment of .beta.-galactosidase used in CEDIA assays) as the labeled analyte. Dworschack, R. T., Rosman, D. B., Shindelman, J. E., Lingenfelter, D. S., and Khanna, P. L., "CEDIA B.sub.12 :A Homogeneous Enzyme-Based Ligand Binding Assay for Serum Vitamin B.sub.12," 15th National Meeting of the Clinical Ligand Assay Society (Los Angeles; May 1989). Also in 1989, Klukas, et. al. presented a chemiluminescent B.sub.12 immunoassay, utilizing intrinsic factor bound to magnetic particles and a B.sub.12 -acridinium ester tracer. Klukas, C., Williams, M., Berg, M., Kozel, P., and Hudson, T., "A Chemiluminescence Receptor Assay for Vitamin B.sub.12," Clin. Chem. 35:1194 (1989). In July 1990, Kuemmerle presented another version of a non-isotopic B.sub.12 EIA at an AACC meeting in San Francisco. The assay employs intrinsic factor bound to a polymeric microspherical solid phase and a B.sub.12 derivative coupled to a reporter enzyme. Kuemmerle, S. C., Boltinghouse, G. L., Delby, S. M., Lane, T. L., Simondsen, R. P., "IMx Assay for Vitamin B.sub.12 ," Clin. Chem. 36:969 (1990).
Of further possible interest are an International Patent Application published under the Patent Cooperation Treaty, Oh, C. S., et. al. (Beckman Instruments, Inc.) WO 89/12826, publication date December 28, 1989; and a published European Patent Application, Hoyle, et. al., EP 0378197A2, published Oct. 1, 1990, both for B.sub.12 non-isotopic assays. The Oh document describes a B.sub.12 EIA using biotinylated intrinsic factor, avidin-horseradish peroxidase and B.sub.12 bound to a solid phase. The Hoyle document contains a B.sub.12 EIA using streptavidin bound to a solid phase, biotinylated monoclonal anti-B.sub.12 and B.sub.12 -horseradish peroxidase.
Also of possible relevance to this invention is an amplification method, recently reported by Self, C. H., EP 0027036B1, October 1988, which amplifies the signal of the labeling enzyme (alkaline phosphatase) approximately 100-fold.
A further document of possible relevance is an investigation of a solid phase system for ELISA assays where streptavidin is bound to a biotinylated protein which in turn is adsorbed onto a microtiter plate, as reported by Suter, M., Butler, J. E., and Peterman, J. H., in "The Immunochemistry of Sandwich ELISAs-III. The Stoichiometry and Efficacy of the Protein-Avidin-Biotin Capture (PABC) System," Molecular Immunoo. 26:221-230 (1989).
Among these methods of the prior art, various difficulties and deficiencies have been noted. The "no-boil" pretreatment method of Allen as referred to above, for example, utilizes treatment reagents which have the potential of denaturing and otherwise detrimentally modifying the various enzymes and binding proteins which might be used in the assay. This will lower the accuracy and reproducilibity of the assay. Also, the amplification method of Self referred to above has not been applied to B.sub.12 assays, since it requires the use of an enzyme-labelled B.sub.12 , which has a much lower binding affinity for proteins than does unlabelled B.sub.12 This has interfered with the development of competitive binding assays in particular for B.sub.12. A still further difficulty arises from the fact that the ability of intrinsic factor to bind to B.sub.12 involves a conformational change in intrinsic factor, and the ability of intrinsic factor to undergo such a change varies with the operating conditions. For maximum binding activity, the intrinsic factor must be in solution. The present invention addresses these and other disadvantages of prior art methods.